Inoculum and process of preparing and using same



Patented Nov. 23, 1948 INOCULUM AND PROCESS OF PREPARING AND USING SAMEFloyd D. Huger, Pearl River, N. Y., assignor, by mesne assignments, toAmerican Cyanamld Company, New York, N. Y., a corporationof Maine NoDrawing. Application May 20, 1944, Serial No. 536,609 8 Claim. (Cl.195-56) This invention relates to a new inoculum and to a process ofpreparing and using the same. More particularly, the invention relatesto the preparation and use of a new kind of inoculum which has beenfound to be useful in the inoculation of nutrient growth media withsurfacegrowing fungi, bacteria, or other microorganisms and sporesthereof.

Certain fermentation processes which are carried out on a commercialscale employ surfacegrowing microorganisms. Fermentation processes ofthis type are usually started by seeding a suitable sterile, liquidgrowth medium with the desired microorganisms or, more often, theirspores. The success of these processes depends in considerable measureupon obtaining a uniform and evenly distributed growth of the organismupon the surface of its growth medium. Since most surface-growingorganisms are aerobic in nature, it is essential that the seed organismsbe distributed on the surface of the growth medium and not below thesurface. It is also essential that the seed organisms be uniformlydistributed so that there are no bare spots.

Ordinarily, large-scale fermentations are started by transferring viablespores, or the organism itself, from the surface of a culture growth,such as an agar slant, to the growth medium. Transfer of the spores by aconventional laboratory inoculating needle is slow. wasteful of spores,and impractical from a com- 'mercial point of view. When it isunderstood that most spores are extremely small, and a small fraction ofa milligram of spore material can seed many square feet of surface area,the difliculties of the problem of obtaining uniform distribution of thespores over the growth surface can be appreciated. Many types of sporesare not readily wet by water and have a tendency to clump, giving veryuneven distribution. Many of those that are thoroughly wet sink and donot grow.

It has been proposed heretofore to transfer spores from a surfaceculture to a carrier substance, such as bran, chaff, or sawdust, andthen introduce this latter spore-bearing material to the growth medium.While such technique speeds up the inoculation process, the use ofmaterials of this type has a number of serious disadvantages. By nature,materials of the type just mentioned are unclean and present a con stantsource of danger through introduction of foreign organisms, or othercontaminants, into the fermentation process. It has also been found thatthese materials tend to become waterlogged and sink in a liquid growthmedium, carrying with them the spores and the material growing thereon.Once below the surface of the liquid, the growin organisms cannot obtaintheir requirements of air, and growth is stopped. Inocula of this typealso absorb substantial quantitles of the fermentation liquor and eitherdecrease the yield of fermentation product or necessitate extractionthereof to obtain the valuable principals of the fermentation liquor.

The present invention is based upon my discovery that conventionalmethods of transferring spores and other viable organisms from onegrowth medium to another can be greatly improved by using a new kind ofinoculum having as a. spore carrier certain substances having propertieswhich will be fully described hereinafter. The spore-carrying mediumwhich may make up a large proportion of the inoculum of the presentinvention is a material which can stand sterilization temperatures, 1.e., C. or higher, without melting or decomposing. It is non-poisonousand does not retard or otherwise interfere with the growth of theorganism to be grown. It is also desirable that the spore-transfermaterial have the ability to pick up and hold viable spores whiletransferring them from the culture to the growth medium.

The preferred transfer material is also waterinsoluble, or at leastslowly soluble, and should have the property of floating whendistributed over the surface of the growth medium. This does notnecessarilymean that the spore-bearing material shall have a specificgravity less than that of the culture medium, since the surface tensionof the nutrient liquor is suflicient in many cases to hold thespore-bearing material on'the surface when deposited thereon. Thespore-bearing material should also have the property of spreading uponthe surface of the growth liquor. It should also be capable of beingpowdered or subdivided to such a small size that it may be dusted orblown in a cloud-like formation over the growth medium. which thensettles uniformly on the surface. It is also desirable, but notnecessary, that the spore-bean ing material be light-colored, so thatits distribution over the surface of the darker growth medium may beobserved. thus enabling the operator to judge the amount and emciency ofthe inoculant on the surface of the growth medium.

Although there are a number of substances which possess the above-namedproperties and which are therefore qualified to act as carriers for theseeding microorganisms, in accordance has been found to be highlysatisfactory. and useful for this purpose. This latter compound ispractically white; it may be easily powdered and then sterilized withoutfusing. It readily picks up seed spores and holds them while inocula--tion is taking place. The spore-bearing, powdered calcium stearate canbe blown or otherwise distributed over the surface of a liquid growthsubstrate. Upon falling on the surface of the liquid, the powderedcalcium stearate rapidly distributes itself uniformly over the surfacethereof. When it is in a powdered form, practically none of it sinks,thereby carrying spores below the surface of the liquid. It iswater-insoluble and does not dissolve in the nutrient liquor. It doesnot interfere with the growth of the organisms for which it was used asa transfer medium.

Of course, other alkali metal and alkaline earth metal salts ofsaturated fatty acids having from 12 to 18 carbon atoms inclusive, whichhave melting points above sterilizing temperatures, about 115 C., mayalso be used. Among these are the calcium, barium, lithium. potassium,and sodium salts of myristic acid, palmitic acid, lauric acid, andstearic acid. The magnesium. potassium, and sodium salts of some ofthese acids, particularly the technical grades, have melting points thatare below some sterilizing temperatures but can be used when absolutelysterile conditions are not necessary. These salts are also morewater-solu ble than the calcium and barium salts but other wise possessmost of the necessary properties for preparing my new inoculum.

In preparing and using my new inoculum, the desired fatty acid soap isfinely divided and then sterilized at temperatures depending upon itssubsequent use. For example, technical calcium stearate is autoclaved at115-120 C. for a suflicient time to sterilize the product, the length oftime required being governed by the size and kind of container. Mosttechnical grades fuse at lower temperatures than the pure salt, and itis necessary to use a product sufllciently pure to withstand sterilizingtemperatures without melting. The impurities in most cases are the saltsof other fatty acids and, except for causing a change of melting point,are as useful as calcium stearate itself. Other chemical or physicalmeans of sterilizing the powder may be used, providing that thetreatment does not chemically or physically mod- .ify the nature of thepowdered salt to such an extent as to make it unsatisfactory for itsintended purpose.

To transfer viable spores, or organisms, to the powdered carrier, Imerely place a quantity of the 7 dry, sterilized powder on the surfacegrowth and agitate the powder to transfer the spores, or otherorganisms, to the powdered material. To expedite this I frequently addto the powdered material insert solid objects of from about 1 of an inchto /2 inch in diameter. Glass beads, pebbles, metal particles, etc.,have been found to be satisfactory. These objects help materially indislodging the spores and relocating them on the fatty acid saltparticles. After the surface-growing spore-bearing material has beenagitated with the powdered salt and glass beads, etc., the latter areremoved by screening, and the powdered inoculum is ready for use.

4 To inoculate a nutrient growth medium with my new inoculum, I usuallydust, by means of any 'I one of several types of dusting devices, aquantity of the spore-bearing powder over the surface of v the nutrientgrowth liquor. The dusted powder settles uniformly over the surface ofthe liquid. and growth of the microorganism is then allowed to takeplace. The growing of the organism is conducted in the usual manner, thefatty acid salt having no noticeable effect upon the normal course ofthe organism's development under otherwise analogous conditions. Sincethe fatty add salt particles are relatively cheap and as they do notabsorb any substantial quantity of the fermentation liquor, there is nonecessity of recovering them.

' Inocula of the type described herein are particularly useful inconjunction with fermentation processes involving the transfer of sporesof surface-growing molds, particularly those of the familyAspergillaceae which includes the genera Penicillium and Asperyillus. Myinoculum has been used very successfully in seeding nutrient growthliquors with spores of Penicillium notatum. The present invention isalso adapted to transfer other surface-growing fungi, bacteria, andyeast cells, or spores of such, to suitable solid or liquid growthmedia. Suitable inocula may be prepared from surface growths in themanner described hereinabove.

It will be understood, of course, that inert material, or even foodsubstances for the organism, may be added to the inoculum withoutdeparting from the essential and novel features thereof as set forth inthe appended claims.

I claim:

1. A dry powdered inoculum comprising a finely-divided saturated fattyacid salt of the group consisting of alkali metal and alkaline earthmetal salts of saturated fatty acids having from 12 to 18 carbon atomsinclusive, said salt having a melting point of at least 120 0., andviable spores of a single species of amold in contact therewith.

2. A dry powdered inoculum comprising finelydivided sterile calciumstearate in intimate con tact with viable spores of Penicillium notatum.

3. The method of preparing an inoculum which comprises sterilizing byheating to a. temperature of at least C. without melting afinely-divided compound of the group consisting of alkali metal andalkaline earth metal salts of saturated fatty acids having from 12'to 18carbon atoms inclusive. said compound having a melting point of at leastC., and agitating said finely-divided sterilized fatty acid salt withthe spores of a surface-growing mold to obtain an inoculum comprising afinely-divided fatty acid salt having viable spores of a single moldspecies deposited on the particles thereof.

4. A methodof preparing an inoculum which comprises contactingsterilized, finely-divided calcium stearate with the viable spores ofPenicillium notatum.

5. A method of inoculating a nutrient growth medium with a mold sporewhich comprises the steps of agitating, a sterile finely-dividedcompound of the group consisting of alkali metal and alkaline earthmetal salts of saturated fatty acids having from 12 to 18 carbon atomsinclusive said compound having a melting point of at least 120 C. over asurface having viable mold spores of a single species thereon to contact.the spores with the said particles and thereafter dusting the mold '6UNITED STATES PATENTS Number Name Date 525,822 Takamine Sept. 11, 18941,166,387 Ramage Dec. 28, 1915 1,260,899 Harris Mar. 26, 1918 1,608,390Holland Nov, 23, 1926 1,833,716 Kluyver Nov. 24, 1931 1,891,750 CornellDec. 20, 1932 2,033,326 Clark Mar. 10, 1936 2,083,065 Heyl June 8, 19372,376,333 Ark May 22, 1945 OTHER REFERENCES Rabbeno, Chemical Abstracts,vol. 22, 1928, page consisting of those of the species Peniclllium 151395 notatum only.

FLOYD D. HAGER.

REFERENCES CITED The following references are of file of this patent:

record in the 20 Coghill, Progress Report No. 14, Sept. 6, 1943, pages 1to 3. Distributed by Committee on Medical Research, 0. B .R.D.

